Exponential fluorescent amplification of individual RNAs using clampFISH probes

Abstract

Abstract:Non-enzymatic, high-gain signal amplification methods with single-cell, single-molecule resolution are in great need. We present a new method (click-amplifying FISH; clampFISH) for the fluorescent detection of RNA that combines the specificity of oligonucleotides with bioorthogonal click chemistry in order to achieve high specificity and high-gain (>400x) signal amplification. We show that clampFISH signal enables detection with low magnification microscopy and separation of cells by RNA levels via flow cytometry. Additionally, we show that clampFISH is multiplexable, compatible with expansion microscopy, and works in tissue samples. Main text:Single molecule RNA fluorescence in situ hybridization (RNA FISH), which enables the direct detection of individual RNA molecules 1,2,3 , has emerged as a powerful technique for measuring both RNA abundance and localization in single cells. Yet, while single molecule RNA FISH is simple and robust, the total signal generated by single molecule RNA FISH probes is relatively low, thus requiring high-magnification microscopy for detection. This keeps the assay relatively low throughput and precludes the ability to combine it with flow cytometry. As such, high efficiency, high gain amplification methods for single molecule RNA FISH signal could enable a host of new applications for RNA FISH.A number of different signal amplification techniques are available for RNA FISH, but each suffers from particular limitations. Approaches such as tyramide signal amplification (TSA) 4 , or enzyme ligated fluorescence (ELF) 5 utilize enzymes to catalyze the deposition of fluorescent substrates near the probes. Alternatively, enzymes can catalyze a "rolling circle" nucleic acid amplification to generate a repeating sequence that can subsequently detected using fluorescent probes [6][7][8] . These methods can lead to large gains in fluorescence, but can suffer from poor sensitivity because of the difficulties in getting bulky enzymes through the fixed cellular environment to the target molecule. Meanwhile, there are a number of non-enzymatic amplification methods, most notably the hybridization chain reaction 9-11 and branched DNA methods [12][13][14] . These methods rely only on hybridization to amplify signal by creating larger DNA scaffolds to which fluorescent probes can attach. However, these methods have an inherent tradeoff between stringency of hybridization and wash conditions for specificity and maintaining hybridization to increase signal gain. Thus, our goal was to create a non-enzymatic, exponential . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/222794 doi: bioRxiv preprint first posted online Nov. 21, 2017; amplification scheme with high sensitivity (detection efficiency), gain (signal amplification), and specificity (low background).We first designed probes that would bind with high specificity and sensitivity; specifically, we ...

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